Summary Long-term alterations in gene expression programs are believed to be key to the development and progression of alcohol use disorder (AUD). The methyl CpG binding protein 2 (MeCP2), the causative gene of Rett syndrome, is a protein that binds methylated DNA and, in turn, recruits transcriptional repressors resulting in persistent down-regulation of gene expression. We observed that MeCP2 mutant mice with reduced capacity to recruit transcriptional repressors exhibit a robust alcohol?related phenotype characterized by heightened sensitivity to the sedative effects of alcohol, reduced alcohol intake in limited access 2-bottle choice and lack of escalation of drinking after passive induction of dependence. Recent evidence indicates that MeCP2's primary function is to recruit a transcriptional repressor complex at sites of methylated DNA through a discrete molecular domain. Importantly, MeCP2 has been found to regulate a specific set of genes ? or regulon ? rather than broadly affecting gene expression levels, and we found significant overlap between alcohol-regulated and MeCP2-regulated genes. Thus, in the present project we will test the overarching hypothesis that MeCP2-regulated genes are key to alcohol's effects and to the transition to escalated alcohol drinking in the setting of alcohol dependence. To test the sub-hypothesis that recruitment of transcriptional repressors by MeCP2 is central to its effects on drinking, we will use MeCP2 mutant mice with reduced capacity to recruit transcriptional repressors in comparison with recently introduced MeCP2 mutant mice with increased capacity to recruit transcriptional repressors, to provide optimal perturbation of MeCP2 function for the analysis of MeCP2 regulated gene networks. To test the sub-hypothesis that specific MeCP2 target genes and modulators are key to the transition to escalated drinking associated with alcohol dependence, we will use a state of the art systems biology strategy that we recently validated for the reconstruction and interrogation of genome-wide transcriptional interactomes from brain gene expression profiles. This approach is centered on unbiased identification of transcriptional regulatory relationships from the gene expression effects of the perturbations under study, rather than what is known from the literature or under different sets of perturbations. Rather than identifying long lists of differentially expressed genes, this systems biology strategy identifies and ranks a small number of genes driving the gene signatures associated with the phenotype. Thus, specific mechanistic hypotheses on the role of MeCP2 in the effects of alcohol are obtained, and will then be experimentally validated in paradigms of dependent and non-dependent alcohol drinking. Ultimately, the results of this study will advance our understanding of the molecular mechanisms behind excessive alcohol drinking in the setting of dependence and will lay the rationale for the exploitation of specific MeCP2-regulated genes and modulators for the development of novel therapeutic concepts for AUD.